Leucine22 -calcitonin

ABSTRACT

Two new peptides are disclosed which have biological activity of the same type as known calcitonins and which are substitution analogs of natural calcitonins. Also resin peptides are disclosed which may be converted to peptides having such biological activity; and processes for producing said resin peptides and said calcitonin peptides.

FIELD OF THE INVENTION

This invention relates to calcitonins having biological activity and topeptides which can be converted to biologically active calcitonins andto processes for preparing such peptides and calcitonins.

BACKGROUND OF THE INVENTION

All known natural calcitonin peptides contain an amino acid sequence of32 amino acids. Salmon calcitonin, for example, has the followingformula: ##STR1##

In U.S. Pat. Nos. 3,926,938, 4,062,815, 3,929,758, 4,033,940 and4,217,268 are disclosed improved syntheses of calcitonins including thesalmon calcitonin above referred to.

The natural calcitonins include the salmon calcitonins, eel, bovine,porcine and ovine calcitonins and human calcitonins. All of these haveeither tyrosine or phenylalanine at position number 22.

SUMMARY OF THE INVENTION

We have discovered synthetic calcitonin peptides with 32 amino acidsthat have biological activity of the same type as known calcitonins. Asignificant difference in structure is that in our new peptides theamino acid sequence does not contain the tyrosine or phenylalanineresidue at position 22 of the known calcitonin amino acid sequence. Thisnew peptide has a leucine at position 22 and is termed Leu 22calcitonin, using the IUPAC-IUB method of nomenclature for syntheticmodifications of peptides [Biochim. Biophys. Acta, 133, 1-5 (1967)]. Thenew peptides have good potency and quality when compared to knowncalcitonins.

DESCRIPTION OF INVENTION

In general we use a solid phase type of synthesis and start with a resincalled benzhydryl amine resin (BHA resin). This resin is derived from across-linked polystyrene bead resin manufactured by copolymerization ofstyrene and divinylbenzene. Resin of this type is known and itspreparation is further demonstrated by Pietta et al [Pietta, P.S. andMarshall, G. R., Chem. Commun., 650 (1970)]. This cross-linkedpolystyrene BHA resin is available from chemical supply houses. We usethe designation ##STR2## to represent the BHA resin in which R is thepolystyrene portion of the resin.

Resin Peptide Synthesis

In this synthesis the amino acids are added one at a time to theinsoluble resin until the total peptide sequence has been built up onthe resin. The functional groups of the amino acids are protected byblocking groups. The α-amino group of the amino acids is protected by atertiary butyloxycarbonyl group or an equivalent thereof. The thiolfunction of cysteine may be protected by benzyl or benzyl protectivegroups described above and designated BZ or by an n-alkylthio group suchas methylthio, ethylthio, n-propylthio, n-butylthio or equivalentsthereof. We use the character R₂ to represent an n-alkylthio group or BZand the character R₁ to represent BZ when R₂ is n-alkylthio and torepresent n-alkylthio when R₂ is BZ. Alternatively, R₁ may be anothercysteine group and when this is the case R₂ is BZ. The guanidinefunction of arginine may be protected by a nitro group, a tosyl group oran equivalent thereof. We use the character T to represent either anitro group or a tosyl group. The ε-amino function of lysine may beprotected by a benzyloxycarbonyl group or a benzyloxycarbonyl derivativesuch as 2-chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl,3,4-dimethylbenzyloxycarbonyl or equivalent thereof. We use thecharacter V to represent benzyloxycarbonyl group or a benzylcarbonylderivative group. The protective group used on the imidazole nitrogen ofhistidine are the benzyloxycarbonyl group and benzyloxycarbonylderivatives such as described above for lysine and are designated as V.The γ-carboxylic acid group of glutamic acid is protected by a benzyl orbenzyl derivative group such as described for the protection of thehydroxyl function of serine and threonine. These protective groups arerepresented by the character BZ. While all Leu-22 calcitonins arecomtemplated within the scope of the invention, salmon calcitonin willbe used for descriptive purposes in this specification.

Other calcitonins include bovine, porcine, ovine, human and eel.

The formula of our new leu 22-salmon calcitonin having activity of thesame type as known salmon calcitonin may be written as follows: ##STR3##

The formula of our new Leu 22-eel calcitonin may be written as follows:##STR4##

As may be seen from the formula above given, 32 amino acids are involvedand in this formula the positions are numbered according to the acceptedprocedure beginning at position 1 for the CYS on one end of the chain,and ending with PRO at position 32 at the other end of the chain. Forclarity of description, this same numbering system will be followed inreferring to the cycles of the synthesis. The assembly of the aminoacids begins with cycle 32 which involves the coupling or proline andcontinues with cycle 31 which involves the coupling of threonine, etc.

Preferred amino acid reactants for use in each of the 32 cycles of thesynthesis are given in the following Table I:

                  TABLE I                                                         ______________________________________                                        Cycle                                                                         Number   Amino Acid Reactant                                                  ______________________________________                                        32       BOC--L--proline                                                      31       BOC--O--benzyl-L--threonine                                          30       BOC--glycine                                                         29       BOC--O--benzyl-L--serine                                             28       BOC--glycine                                                         27       BOC--O--benzyl-L--threonine                                          26       BOC--L--asparagine p-nitrophenyl ester                               25       BOC--O--benzyl-L--threonine                                          24       BOC--Ω-nitro-L--arginine or                                             BOC--Ω-tosyl-L--arginine                                       23       BOC--L--proline                                                      22       BOC--L--Leucine                                                      21       BOC--O--benzyl-L--threonine                                          20       BOC--L--glutamine p-nitrophenyl ester                                19       BOC--L--leucine                                                      18       BOC--ε-CBZ--L--lysine or                                              BOC--ε-2-chlorobenzyloxycarbonyl-L--lysine                   17       BOC--N(im)--CBZ--L--histidine                                        16       BOC--L--leucine                                                      15       BOC--L--glutamic acid γ-benzyl ester                           14       BOC--L--glutamine p-nitrophenyl ester                                13       BOC--L--benzyl-L--serine                                             12       BOC--L--leucine                                                      11       BOC--ε-CBZ--L--lysine or                                              BOC--ε-2-chlorobenzyloxycarbonyl-L--lysine                   10       BOC--glycine                                                         9        BOC--L--leucine                                                      8        BOC--L--valine                                                       7        BOC--S--ethylthio-L--cysteine,                                                BOC--S--methylthio-L--cysteine,                                               BOC--S--n-propylthio-L--cysteine or                                           BOC--S--n-butylthio-L--cysteine                                      6        BOC--O--benzyl-L--threonine                                          5        BOC--O--benzyl-L--serine                                             4        BOC--L--leucine                                                      3        BOC--L--asparagine p-nitrophenyl ester                               2        BOC--O--benzyl-L--serine                                             1        BOC--S--p-methoxybenzyl-L--cysteine,                                          BOC--S--benzyl-L--cysteine or                                                 BOC--S--3,4-dimethylbenzyl-L--cysteine                                        Bis--Boc--Cystine                                                    ______________________________________                                    

Each of the amino acid derivatives mentioned in Table I may be purchasedfrom supply houses.

CYCLE 32 Coupling Of Proline To BHA Resin

The reaction vessel used in all steps of the resin peptide synthesis maybe a glass vessel equipped with inlet ports at the top for addition ofmaterials and a sintered glass disk at the bottom for removal of solublereaction mixtures and wash solvents by filtration. Filtration may beperformed either by vacuum or the use of nitrogen pressure. The contentsof the vessel may be agitated by shaking the entire vessel or bymechanical stirrer.

In cycle 32 the BHA resin is placed in the reaction vessel and suspendedin a solvent such as methylene chloride, chloroform, dimethylformamide,benzene or equivalents thereof in proportions of about 3 to 12 ml. ofsolvent per gram of resin. To this is added BOC-L-proline in an amountof about 1 to 6 equivalents per free amine equivalent of the BHA resinemployed. After a period of mixing of 5 to 10 minutes, a couplingreagent (CA) such as dicyclohexyl carbodiimide (DCC) may be added, orother diimide coupling agents may be used. The diimide coupling agentmay be used in the amount of 0.5 to 2.0 equivalents per equivalent ofBOC-L-proline used.

The BOC-L-proline may be coupled in the absence of a coupling reagent ifits active ester derivative, its azide derivative, its symmetricalanhydride derivative, or a suitably chosen mixed anhydride derivative isused. Active ester derivatives that may be employed are 2-nitrophenylester, 4-nitrophenyl ester, pentafluorophenyl ester, N-hydroxysuccimideester or equivalents thereof. The active esters are used in amounts of 1to 10 equivalents per free amine equivalent of BHA resin.

The reaction mixture consisting of the BHA resin, the solvent, theBOC-L-proline, and the coupling reagent or BOC-L-proline active ester isstirred or shaken mechanically until the reaction is complete asindicated by a ninhydrin test [E. Kaiser, et al., Anal. Biochem., 34,595-8 (1970)] on a test sample. After completion of the couplingreaction, the BOC-L-proline resin may be washed with solvents such asmethylene chloride, chloroform, methyl alcohol, benzene,dimethylformamide, or acetic acid. The amount of wash solvent maysuitably be 5 to 20 ml. of solvent for each gram of BHA resin usedinitially. If it is desired to terminate the coupling reaction beforecompletion, the washing procedure may be used and the remaining freeamino groups on the BOC-L-proline resin may be blocked from furtherreaction by acetylation with an excess of acetylation reagents. Theacetylation procedure may be performed by agitating the BOC-L-prolineresin with a solution of the acetylation reagent for a period of 0.5 to12 hours. Acetylation reagents such as N-acetylimidazole in methylenechloride solution or a mixture of acetic anhydride and triethylamine inchloroform may be used. The acetylation reagent may be used in theamount of 0.5 to 5.0 equivalents per equivalent of free amine titer ofthe starting BHA resin.

The coupling reaction to produce the BOC-L-proline resin may bedescribed by the following formula: ##STR5##

Deprotection Of BOC-L-Proline Resin

The BOC-L-proline resin produced as above described may be washed with asolvent such as referred to above and deprotected by agitating it withan agent such as a mixture of trifluoroacetic acid (TFA) in a solventsuch as methylene chloride, chloroform, benzene or equivalents thereof.The amount of TFA in the solvent may vary from 10 to 100% of themixture. The amount of TFA-solvent mixture may vary from 3 to 20 ml. pergram of BHA resin used initially. The reaction time may be from about 10minutes to 4 hours. The deprotection step is terminated by filtration toremove the TFA-solvent mixture. The residual TFA may be removed from theL-proline resin by washing with 3 to 20 ml. per gram of BHA resin ofsolution of 5 to 30% of triethylamine in a solvent such as methylenechloride, chloroform, benzene or equivalents thereof. Other tertiary orsecondary organic amines may be used in place of the triethylamine, suchas, trimethylamine, N-ethylpiperidine, diisopropylamine or equivalentsthereof. The free amine titer of the L-proline resin may be determinedby the Dorman titration procedure (Dorman, L.C., Tetrahedron Letters,1969, 2319-21). The deprotection reaction may be described by thefollowing formula: ##STR6##

CYCLE 31

The prolyl BHA resin obtained as a result of cycle 32 may be suspendedin a coupling solvent, the BOC-O-BZ-L threonine derivative added and themixture equilibrated in the same manner. The coupling agent, DCC, may beadded, and after completion of the reaction as indicated by theninhydrin test, the reaction mixture may be removed from the BOC-O-BZthreonylpropyl BHA resin by filtration. The peptide resin may be washedwith solvents. The amounts of reactants and solvents and reaction timesmay be the same as described in cycle 32. The BOC group may be removedfrom the peptide resin by the deprotection method described in the cycle32. The resulting O-BZ-threonylprolyl BHA resin is then ready for cycle30. The reactions of the cycle 31 may be shown by the following formula:##STR7##

For convenience, we may write this resulting resin peptide usingabbreviated nomenclature as follows: ##STR8##

CYCLE 30

In cycle 30, the coupling reaction and also the deprotection reactionmay be performed in the same manner as in cycle 31 except thatBOC-glycine is used in place of BOC-O-BZ-L-threonine. The reactionthrough coupling and deprotection may be written: ##STR9##

CYCLE 29

In cycle 30, the coupling and deprotection reactions may be performed inthe same manner as in cycle 31 except for the substitution ofBOC-O-BZ-L-serine as the amino acid derivative. This may be written.##STR10##

CYCLE 28

In cycle 28, the coupling and deprotection reactions are performed asdescribed in cycle 31 except that BOC-glycine is substituted as theamino acid reactant. These reactions through coupling and deprotectionmay be written as follows: ##STR11##

CYCLE 27

In this cycle, the coupling and deprotection reactions may be as incycle 31 using the same amino acid reactant, resulting in the followingcompound: ##STR12##

CYCLE 26

In cycle 26, the coupling reaction is performed using an active esterderivative of BOC-L-asparagine. The active ester procedure is used inplace of the DCC coupling agent with BOC-asparagine or BOC-glutamine.The reaction may be performed using the active ester derivative ofBOC-L-asparagine in the amount of 2 to 10 equivalents per free amineequivalent of BHA resin in dimethylformamide, mixtures ofdimethylformamide with benzene, methylene chloride or chloroform or withequivalents thereof in amount of 2 to 20 ml. of solvent per gram of BHAresin used initially. Reaction times range from 1 to 72 hours. Thereaction mixture may be removed from the BOC peptide resin by filtrationafter completion of the reaction as indicated by a ninhydrin test. Theactive ester derivatives employed may be 2-nitrophenyl esters,4-nitrophenyl esters, pentafluorophenyl, or equivalents thereof. We useAE to designate the active ester portion of the derivative. The couplingreaction may be written: ##STR13##

The deprotection reaction to remove the BOC group is performed as incycle 32.

CYCLES 25-21

In each of cycles 25 to 21, the coupling and deprotection reactions maybe conducted using the methods and proportions of reactants as in cycle31, using BOC-BZ-L-threonine in cycle 25, BOC- -T-L-arginine in cycle24, BOC-L-proline in cycle 23, BOC-L-leucine in cycle 22, andBOC-O-BZ-L-threonine in cycle 21. The compound resulting from thecompletion of cycle 21 may be written. ##STR14##

CYCLE 20

In cycle 20, the coupling and deprotection reactions may be performedusing the methods and proportions of reactants as in cycle 26 using aBOC-L-glutamine active ester derivative as the amino acid derivative,resulting in the compound: ##STR15##

CYCLE 19

In cycle 19, the reactions are performed as in cycle 31 usingBOC-L-leucine as the amino acid derivative. The compound resulting fromcycle 19 is: ##STR16##

CYCLE 18

In cycle 18, we may use as the amino acid derivative BOC-ε-V-L-lysine.Otherwise, cycle 18 methods may be performed as in cycle 31 resulting inthe compound: ##STR17##

CYCLES 17-15

Cycles 17 to 15 may be performed as in cycle 31 except for the use ofBOC-N(im)-V-L-histidine in cycle 17, BOC-L-leucine as the reactant incycle 16 and BOC-L-glutamic acid BZ ester (BZ represents the same groupsas it represents for serine and threonine) as the reactant in cycle 15,resulting in the following compound from cycle 15: ##STR18##

CYCLES 14-8

Cycle 14 may be performed identically to cycle 20 usingBOC-L-glutamine-AE as the amino acid derivative. Cycles 13 to 8 may beperformed as in cycle 31 except for the use of BOC-O-BZ-L-serine incycle 13, BOC-L-leucine in cycle 12, BOC-ε-V-L-lysine in cycle 11,BOC-glycine in cycle 10, BOC-L-leucine in cycle 9, and BOC-L-valine incycle 8 resulting in the compound: ##STR19##

CYCLE 7

Cycle 7 may be performed as in cycle 31 except for the use ofBOC-S-L-cysteine or for the amino acid derivative. The compoundsresulting from cycle 7 are described by the formula: ##STR20## where R₂is an alkylthio or a BZ group.

CYCLES 6-2

Cycles 6 to 4 may be performed as in cycle 31 except thatBOC-O-BZ-L-threonine be used as the amino acid derivative in cycle 6,BOC-BZ-L-serine may be used as the amino acid derivative in cycle 5 andcycle 2, and BOC-L-leucine may be used in cycle 4 as the amino acidderivative. Cycle 3 may be performed identically to cycle 26 usingBOC-L-asparagine active ester. The compound resulting from cycle 2 is:##STR21##

CYCLE 1

This cycle may be performed identically to cycle 7 usingBOC-S-R-L-cysteine derivatives. The R group chosen for the cysteine maybe the same as used in cycle 7 or different. For example, if thederivative chosen for cycle 7 is BOC-S-ethylthio-L-cysteine, thederivative in cycle 1 may be BOC-S-4-methoxybenzyl-L-cysteine or ifBOC-S-4-methoxybenzyl-L-cysteine was chosen for cycle 7, then thisderivative may be used also in cycle 1. The compounds resulting fromcycle 1 are illustrated by the formula: ##STR22## where R₁ is S-n-alkyl,CYS or Bz and R₂ is S-n-alkyl or Bz, R₁ being alkyl or CYS when R₂ is Bzand R₂ being Bz when R₁ is S-n-alkyl or CYS.

Cycle 1 represents the completion of the resin peptide, the resinpeptide may be removed from the reaction vessel and dried in a vacuum.The weight of the resin peptide may be expected to be from 2.0 to 4.0times weight of BHA resin used initially in the synthesis.

Resin Peptide Cleavage

The peptide is cleaved from the resin peptide resulting from cycle 1 bytreatment with liquid hydrogen fluoride (HF). The HF cleavage reactionmay be performed by treating a mixture of the resin peptide and anisole(0.5 to 5 ml. for each gram of resin peptide) with liquid HF (2 to 20ml. for each gram of resin peptide) for 0.5 to 20 hours at -20 degreesto +15 degrees centigrade. After the reaction period, the excess HF maybe removed by evaporation and the resulting mixture of peptide and resinbeads may be extracted with organic solvent such as ethyl acetate,diethyl ether, benzene or the like to remove the anisole and residual ofHF. The peptide may be separated from the resin beads by extraction intoaqueous acetic acid. The peptide at this stage is not cyclic but is thenon-cyclic product without the cyclic disulfide bond between thecysteines at positions 1 and 7 in the molecule.

The HF treatment removes all blocking groups from the peptide, exceptthe S-alkylthio blocking groups on the thiol function of cysteineresidue at either position 1 or 7. The S-n-alkylthio-L-cysteine residueis stable to the HF cleavage procedure and remains intact throughout thecleavage and extraction procedures. The S-BZ-L-cysteine residue iscleaved by HF to yield a cysteine residue with a free thiol function.Both types of blocking groups have been employed during our synthesis incombination with each other at positions 1 and 7.

Thus, the peptides obtained after HF cleavage can be one of three typesdepending upon the blocking groups chosen for the thiol function of thecysteine derivative used during the resin peptide synthesis.

If BOC-S-BZ-L-cysteine derivatives are used in the resin peptidesynthesis cycle 1 and BOC-S-n-alkylthio-L-cysteine is used in cycle 7,the peptide resulting after HF cleavage would be of Type I and wouldhave a free thiol function at position 1 and have a S-n-alkylthiofunction on the cystine residue at position 7. We call this a Type Ipeptide which is represented by the formula given as follows: ##STR23##

Conversely, if BOC-S-n-alkylthio-L-cysteine derivative is used in cycle1 and the BOC-S-BZ-L-cysteines were used in position 7, the peptideresulting from the cleavage would be of Type II and would be representedby the formula: ##STR24##

In place of the protecting group S-n-alkyl at position 1 we may use acysteine group (which with the cysteine at this position forms a cystinegroup) and when we do this we use a BZ group for protecting the cysteineat position 7. If Bis-BOC-L-cysteine is used as the reactant in cycle 1and the BOC-S-BZ-L-cysteine is used as the reactant in cycle 7, thepeptide resulting from the cleavage will be of Type III and may berepresented by the following formula: ##STR25##

If BOC-BZ-L-cysteine is used as the reactant in both positions 1 and 7,the peptide resulting from the cleavage will be of Type IV andrepresented by the following formula:

    H-CYS-SER-ASN-LEU-SER-THR-CYS-VAL-LEU-GLY-LYS-LEU-SER-GLN-GLU-LEU-HIS-LYS-LEU-GLN-THR-LEU-PRO-ARG-THR-ASN-THR-GLY-SER-GLY-THR-PRO-NH.sub.2

The conversion of Types I, II and III peptides to the cyclic disulfidepeptides may be performed by diluting with distilled water the aqueousacetic acid solution of the crude peptides from HF cleavage to a finalvolume of 50 to 200 ml. per gram of resin peptide cleaved. The pH ofthis solution may be adjusted to 5 to 10 by the addition of ammoniumhydroxide solution and the mixture may be stirred in a closed containerunder a stream of an inert gas such as nitrogen for about 2 to 48 hours.The reaction period can be stopped when the off-gas stream no longercontains n-alkylmercaptan. The pH of the reaction mixture may be loweredto about 3.5 to 5.5 by the addition of glacial acetic acid.

The conversion to Type IV peptides to the cyclic disulfide peptide maybe performed by the classical method known to the art in which thepeptides are oxidized to join the ring structure to include in the ringthe cysteines at positions 1 and 7.

Whether the intermediate peptides are of Type I, II, III or IV we maysynthesize peptides having amino acid chains corresponding to any knowcalcitonin except for the substitution of leucine at position 22, andsuch peptide synthesized as herein set forth may be purified and foundto have the same type of biological activity as this known calcitonin.Any calcitonin so synthesized is designated Leu 22-calcitonin. This isin accordance with the IUPAC-IUB method of nomenclature.

Purification Of The Crude Leu 22-SCT

The crude peptide solutions at pH 5.0 from the above synthesis may beconcentrated using an ion-exchange procedure. The concentrate may bepurified by a combination of gel-filtration procedures and ion-exchangechromatography methods. The final purified product may be obtained fromsolution by freeze-drying as a fluffy white solid. The product gives thecorrect amino acid analysis for the desired peptide.

Following is the specific example of the preparation of the peptide.

EXAMPLE 1 Resin Activation

The BHA resin (5 g.) with an amine titer of 0.61 meq./g. was placed inthe reactor vessel of a peptide synthesizer marketed by Schwarz-Mann,Inc. of Orangeburg, New York. The resin was treated with 25 ml. of thefollowing solvents filtering after each treatment:

Methylene chloride for 2 minutes

Chloroform for 2 minutes two times each

10% triethylamine in chloroform for 5 minutes two times each

Chloroform for 2 minutes

Methylene chloride for 2 minutes three times each

Cycle 32

Coupling: The BHA resin, 25 ml. of methylene chloride and 2.58 g. (0.012moles) of BOC-L-proline was stirred for 10 minutes. 12.0 ml. ofmethylene chloride solution of dicyclohexylcarbodiimide (1milliequivalent of DCCI per 1 ml. of solution) was added to the reactorand the mixture agitated for 6 hours. The reaction mixture was removedfrom the reactor by filtration and the BOC-prolyl BHA resin subjected tothe following successive 2 minute, 25 ml. washes, removing the wash byfiltration each time:

Methylene chloride two times

Methyl alcohol two times

Methylene chloride three times

Acetylation: The resin was then agitated with a mixture of 1.5 ml. oftriethylamine (TEA), 1 ml. of acetic anhydride and 25 ml. of chloroformfor 2 hours. The reaction mixture was removed by filtration and theresin subjected to the following 2 minute, 25 ml. washes:

Chloroform two times

Methyl alcohol two times

Methylene chloride three times

Deprotection: The BOC-protected resin was agitated for 5 minutes with amixture of 15 ml. of trifluoroacetic acid (TFA) and 15 ml. of methylenechloride. This mixture was removed by filtration and the resin wasagitated with a second mixture of 15 ml. of TFA and 15 ml. of methylenechloride for 30 minutes. The reaction mixture was removed by filtrationand the resin subject to the following 25 ml. washes:

Methylene chloride two times two minutes each

Methyl alcohol two times two minutes each

Chloroform two times two minutes each

10% TFA in chloroform two times ten minutes each

Chloroform two times two minutes each

Methylene chloride two times two minutes each

The L-proline BHA resin was titrated to establish the amine or prolinetiter. This value was 0.55 milliequivalents of amine or proline per gramof resin.

Cycle 31

Coupling: The L-prolyl resin, 25 ml. of methylene chloride and 3.4 g.(0.011 mole) of BOC-O-benzyl-L-threonine were agitated for 10 minutes.Then 11.0 ml. of methylene chloride solution of dicyclohexylcarbodiimide(1 milliequivalent of DCCI per 1 ml. of solution or a total of 0.011mole of DCCI) was added to the reactor and the mixture agitated for 2hours. The reaction mixture was removed from the reactor and the resinwas subjected to the following successive 2 minute, 25 ml. washes,removing the wash by filtration each time.

Methylene chloride two times

Methyl alcohol two times

Methylene chloride three times

A ninhydrin test was negative.

Deprotection: The deprotection procedure described in cycle 32 wasrepeated for this cycle.

Cycles 30 Through 27

The coupling and deprotection procedures used in these cycles were thesame as in cycle 31 except that the following amino acid derivativeswere used in place of the threonine derivative:

Cycle 30--1.92 g. (0.011 mole) of BOC glycine

Cycle 29--3.25 g. (0.011 mole) of BOC-O-benzyl-L-serine

Cycle 28--The reactant used was the same as cycle 30

Cycle 27--The reactant used was the same as cycle 31

Cycle 26

Coupling: The peptide resin obtained from cycle 27 was washed twice with25 ml. portions of dimethylformamide (DMF). The resin was then agitatedfor 24 hours with a solution of 5.82 g. (0.0165 mole) ofBOC-L-asparagine p-nitrophenyl ester in 35 ml. of DMF. The reactionmixture was filtered and the resin peptide subjected to two minutewashes with two successive 25 ml. portions of the following solvents:DMF, methylene chloride, methanol, methylene chloride. Each individualsolvent was removed by filtration. A ninhydrin test was negative.

Deprotection: The deprotection procedure used in cycle 32 was repeated.

Cycle 25

Coupling and deprotection procedures were the same as in cycle 31 usingthe same reactants and amounts.

Cycle 24

Coupling: The resin peptide obtained from cycle 25 was washed with twosuccessive 25 ml. portions of DMF. The resin peptide was then agitatedfor 10 minutes with a mixture of 7.06 mg. (0.0165 mole) ofBOC-N-y-tosyl-L-arginine and 25 ml. of DMF. Then 16.5 ml. of DCCI inmethylene chloride (equivalent to 0.0165 mole of DCCI) was added and themixture agitated for 6 hours. The reaction mixture was removed byfiltration. The resin peptide was subjected to two minute washes withtwo successive 25 ml. portions of the following solvents: DMF, methylenechloride, methyl alcohol, methylene chloride. The ninhydrin test wasnegative.

Deprotection: The deprotection used in cycle 32 was repeated.

Cycle 23

Coupling: The peptide resin obtained from cycle 24 was agitated for 10minutes with 3.54 g. (0.0165 mole) of BOC-L-proline and 25 ml. ofmethylene chloride. 16.5 ml. of DCCI in methylene chloride (equivalentto 0.0165 mole of DCCI) was added and the mixture agitated for 6 hours.The reaction mixture was removed by filtration and the resin peptidesubjected to two minute washes with two successive 25 ml. portions ofthe following solvents: methylene chloride, methyl alcohol, methylenechloride. Each individual wash was removed by filtration. The ninhydrintest was negative.

Deprotection: The deprotection used in cycle 32 was repeated.

Cycles 22 and 21

The coupling and deprotection procedures used in these cycles were thesame as in cycle 23 except that in the coupling reaction the followingamino acid derivative was used in place of BOC-L-proline.

Cycle 22--4.90 g. (0.022 mole) BOC-L-leucine

Cycle 21--5.1 g. (0.0165 mole) of BOC-O-benzyl-L-threonine

Cycle 20

This procedure is the same as cycle 26 except that 6.05 g. (0.0165 mole)of BOC-L-glutamine p-nitrophenyl ester is used in place of theasparagine derivative.

Cycles 19 through 15

The procedure is the same as used in cycle 31 except that the followingamino acid derivatives were used in place of the threonine derivative:

Cycle 19--2.54 g. (0.011 mole) of BOC-L-leucine

Cycle 18--4.55 g. (0.011 mole) of BOC-ε-2-chlorocarbobenzyloxy-L-lysine

Cycle 17--5.14 g. (0.011 mole) of BOC-N(im)-carbobenzyloxy-L-histidine

Cycle 16--See cycle 19

Cycle 15--3.70 g. (0.011 mole) of BOC-L-glutamic acid-γ-benzyl ester

Cycle 14

Same as cycle 20.

Cycle 13

The procedure used was the same as was used in cycle 23 except that inthe coupling reaction 4.86 g. (0.0165 mole) of BOC-O-benzyl-L-serine wasused in place of the proline derivative.

Cycles 12 through 9

The procedures used were the same as used in cycle 31 except in thecoupling reactions the following amino acid derivatives were used inplace of the threonine derivative.

Cycle 12--Same reactants as used in cycle 19

Cycle 11--The reactants were the same as in cycle 18

Cycle 10--Same reactants as used in cycle 30

Cycle 9--Same reactants as used in cycle 19

Cycle 6

Coupling: The resin peptide from cycle 9 was agitated for 10 minuteswith 3.85 g. (0.0165 mole) of BOC-L-valine and 25 ml. of methylenechloride. Then 16.5 ml. of DCCI in methylene chloride (equivalent to0.0165 mole of DCCI) was added and the mixture agitated for 16 hours.The reaction mixture was removed by filtration. The resin peptide wassubjected to two minute washes with two successive 25 ml. portions ofthe following solvents: methylene chloride, methyl alcohol, methylchloride. Each individual wash was removed by filtration.

Deprotection: See cycle 31.

Cycle 7

The procedure was the same as used in cycle 31 except that in thecoupling reaction 3.09 g. (0.011) of BOC-S-ethylthio-L-cysteine was usedin place of the threonine derivative.

Cycle 6

The reactants and procedures used were the same as cycle 31.

Cycle 5

The reactants and procedures used were the same as cycle 29.

Cycle 4

The reactants and procedures used were the same as cycle 19.

Cycle 3

The reactants and procedures used were the same as cycle 26.

Cycle 2

The reactants and procedures used were the same as cycle 29.

Cycle 1

The reactants and procedures used were the same as cycle 31 except that3.75 g. (0.011 mole) of BOC-S-p-methoxybenzyl-L-cysteine was used inplace of the threonine derivative.

After completion of cycle 1, the resin peptide was washed with twosuccessive 25 ml. portion of n-hexane. The peptide material was removedfrom the reactor and dried in an electric vacuum oven at 40 degreescentigrade and 0.1 mm. of Hg for 24 hours.

Cleavage With Hydrogen Fluoride

The dried resin peptide (2 g.) and 2 ml. of anisole were placed in aTeflon reaction vessel. The vessel equipped with a Teflon-coated magnetstirrer was placed in a dry ice-acetone bath and 10 ml. of hydrogenfluoride gas was condensed into the vessel. This mixture was stirred at0 degrees centigrade in an ice bath for 1 hour. The hydrogen fluoridewas removed by evaporation at reduced pressure. The residue wastriturated with six 10 ml. portions of ethyl acetate. The peptide wasextracted from the resin beads with 120 ml. of 0.1 molar aqueous aceticsolution.

Cyclization Of The Peptide

The aqueous acetic acid extract obtained from hydrogen fluoride cleavagewas diluted to 0.2 liters by addition of 80 ml. of distilled water. ThepH of the solution was adjusted to 7.5 by the addition of concentratedammonium hydroxide. The solution was stirred in a closed vessel under astream of nitrogen for 24 hours. At this time no ethyl mercaptan couldbe detected in the emerging nitrogen stream. The ethyl mercaptan contentof the nitrogen stream was measured by passing the stream through asolution of Ellman's reagent [Ellman, G. L., Arch. Biochem. Biophys.,82, 70-7, (1969)]. The pH of the reaction mixture was adjusted to 5.0 byaddition of glacial acetic acid.

Purification Of The Crude Leu 22-SCT

The 0.2 liters of solution from the above synthesis at pH 5.0 wasconcentrated using a SP-Sephadex C-25 ion-exchange column. The 25 ml.concentrate removed from the column with 0.7 molar sodium chloridesolution was desalted and purified by passing through a Sephadex G-25(fine) gel-filtration column and eluting with 0.03 molar aqueous aceticacid solution. The Leu 22-SCT fraction from this column was adjusted topH 6.0 by addition of ammonium hydroxide solution. This solution wasfurther purified by ion-exchange chromatography using a Whatman CM52column eluted with ammonium acetate buffer. The Leu 22-SCT fraction fromthis column was adjusted to pH 5.0 by addition of glacial acetic acid.This solution was concentrated using a SP-Sephadex C-25 ion-exchangecolumn. The 30 ml. concentrate removed from the column with 0.7 molarsodium chloride solution was desalted with a Sephadex G-25 (fine)gel-filtration column. The purified peptide fraction was collected andfreeze-dried. The product was obtained as a fluffy white solid. Aminoacid analysis of the product gave the following ratios with thetheoretical values given in parenthesis: Asp 2.0 (2), Thr 5.0 (5), Ser3.9 (4), Glu 3.3 (3), Pro 2.2 (2), Gly 2.8 (3), Val 1.0 (1), Leu 6.0(6), His 0.9 (1), Lys 1.9 (2), Arg 0.8 (1), Cys 1.85 (2). This productassayed at 3800 MRC units per mg.

While only certain embodiments of our invention have been described inspecific detail it will be apparent to those skilled in this art thatmany other specific embodiments may be practiced and many changes may bemade, all within the spirit of the invention and the scope of theappended claims.

What is claimed:
 1. A peptide having the structure: ##STR26##
 2. Apeptide having the structure: ##STR27##
 3. A peptide having thestructure: ##STR28## where R₁ is S-n-alkyl, Cys or H and R₂ is S-n-alkylor H, R₁ being S-n-alkyl, Cys or H when R₂ is H and R₂ being S-n-alkylor H when R₁ is H and H when R₁ is S-n-alkyl or Cys, wherein S-n-alkylis selected from the group consisting of methylthio, ethylthio,n-propylthio, n-butylthio, and equivalents thereof.
 4. A peptide havingthe structure: ##STR29## where R₁ is S-n-alkyl, Cys or H and R₂ isS-n-alkyl or H, R₁ being S-n-alkyl, Cys or H when R₂ is H and R₂ beingS-n-alkyl or H when R₁ is H and H when R₁ is S-n-alkyl or Cys, whereinS-n-alkyl is selected from the group consisting of methylthio,ethylthio, n-propylthio, n-butylthio, and equivalents thereof.
 5. ALeu-22 calcitonin selected from the group consisting of Leu-22 salmoncalcitonin and Leu-22 eel calcitonin.